Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway

Astaxanthin is a high-value red pigment and antioxidant used by pharmaceutical, cosmetics, and food industries. The astaxanthin produced chemically is costly and is not approved for human consumption due to the presence of by-products. The astaxanthin production by natural microalgae requires large open areas and specialized equipment, the process takes a long time, and results in low titers. Recombinant microbial cell factories can be engineered to produce astaxanthin by fermentation in standard equipment. In this work, an oleaginous yeast Yarrowia lipolytica was engineered to produce astaxanthin at high titers in submerged fermentation. First, a platform strain was created with an optimised pathway towards β-carotene. The platform strain produced 331 ± 66 mg/L of β-carotene in small-scale cultivation, with the cellular content of 2.25% of dry cell weight. Next, the genes encoding β-ketolase and β-hydroxylase of bacterial (Paracoccus sp. and Pantoea ananatis) and algal (Haematococcus pluvialis) origins were introduced into the platform strain in different copy numbers. The resulting strains were screened for astaxanthin production, and the best strain, containing algal β-ketolase and β-hydroxylase, resulted in astaxanthin titer of 44 ± 1 mg/L. The same strain was cultivated in controlled bioreactors, and a titer of 285 ± 19 mg/L of astaxanthin was obtained after seven days of fermentation on complex medium with glucose. Our study shows the potential of Y. lipolytica as the cell factory for astaxanthin production.

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Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699235 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuxiao Xie ◽

Shulin Chen ◽

Xiaochao Xiong

Keyword(s):

Yarrowia Lipolytica ◽

Fold Increase ◽

Genetically Engineered ◽

Cell Factory ◽

Yeast Nitrogen Base ◽

Nitrogen Base ◽

Yeast Extract Peptone Dextrose ◽

Dry Cell Weight ◽

A Cell ◽

Β Carotene

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

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Biovalorisation of crude glycerol and xylose into xylitol by oleaginous yeast Yarrowia lipolytica

10.21203/rs.3.rs-19009/v2 ◽

2020 ◽

Author(s):

Ashish Prabhu ◽

Dominic J Thomas ◽

Rodrigo Ledesma- Amaro ◽

Gary A Leeke ◽

Angel Medina Vaya ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Crude Glycerol ◽

Oleaginous Yeast ◽

Scale Up ◽

Microbial Cell ◽

Department Of Energy ◽

Cell Factory ◽

Potential Cost ◽

Cell Factories ◽

Pharmaceutical Industries

Abstract Background: Xylitol is a commercially important chemical with multiple applications in the food and pharmaceutical industries. According to the US Department of Energy, xylitol is one of the top twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is however expensive and energy intensive. In contrast, the biological route using microbial cell factories offers a potential cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts and biomass which can be sourced from renewable carbon originating from a variety of cheap waste feedstocks. Result: In this study, biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica an oleaginous yeast which was firstly grown on a glycerol/glucose for screening of co-substrate, followed by media optimisation in shake flask, scale up in bioreactor and downstream processing of xylitol. A two-step medium optimization was employed using central composite design and artificial neural network coupled with genetic algorithm. The yeast amassed a concentration of 53.2 g/L xylitol using pure glycerol (PG) and xylose with a bioconversion yield of 0.97 g/g. Similar results were obtained when PG was substituted with crude glycerol (CG) from the biodiesel industry (titer: 50.5 g/L; yield: 0.92 g/g). Even when xylose from sugarcane bagasse hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. Xylitol was successfully crystallized from PG/xylose and CG /xylose fermentation broths with a recovery of 39.5 and 35.3%, respectively. Conclusion: To the best of the author’s knowledge, this study demonstrates for the first time the potential of using Y. lipolytica as a microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.

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Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

Applied and Environmental Microbiology ◽

10.1128/aem.02079-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7360-7370 ◽

Cited By ~ 54

Author(s):

John Seip ◽

Raymond Jackson ◽

Hongxian He ◽

Quinn Zhu ◽

Seung-Pyo Hong

Keyword(s):

Yarrowia Lipolytica ◽

Lipid Accumulation ◽

Oleaginous Yeast ◽

De Novo ◽

Lipid Synthesis ◽

Omega 3 ◽

Wild Type ◽

Content Type ◽

Reverse Transcription Pcr ◽

Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

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Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Scientific Reports ◽

10.1038/s41598-021-85170-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sivamoke Dissook ◽

Tomohisa Kuzuyama ◽

Yuri Nishimoto ◽

Shigeru Kitani ◽

Sastia Putri ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Isoprenoid Biosynthesis ◽

Mep Pathway ◽

Authentic Standard ◽

Chemical Inhibition ◽

Chromatographic Peaks ◽

Methyl Erythritol Phosphate ◽

Limiting Condition ◽

Yeast Yarrowia Lipolytica

AbstractMethyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

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